EP2318536B1 - Kombination von zwei genetischen elementen zur kontrolle der blütenentwicklung einer zweikeimblättrigen pflanze, deren anwendung bei nachweis- und selektionsverfahren - Google Patents

Kombination von zwei genetischen elementen zur kontrolle der blütenentwicklung einer zweikeimblättrigen pflanze, deren anwendung bei nachweis- und selektionsverfahren Download PDF

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EP2318536B1
EP2318536B1 EP09740394.3A EP09740394A EP2318536B1 EP 2318536 B1 EP2318536 B1 EP 2318536B1 EP 09740394 A EP09740394 A EP 09740394A EP 2318536 B1 EP2318536 B1 EP 2318536B1
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nucleic acid
protein
nucleotide
allele
sequence seq
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EP2318536A2 (de
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Abdelhafid Bendahmane
Adnane Boualem
Christelle Troadec
Martin Antoine
Catherine Dogimont
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Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
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Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to the field of selection of varieties of plants, and in particular to the selection of the sexual type of plants. It relates to the genotypic detection of the sex of plants by analysis of the polymorphism of an A gene and a G gene, as well as to means of implementing this detection and to methods of obtaining plants, the sexual phenotype is changed.
  • hybrid plants thanks to the heterosis phenomenon also called hybrid vigor, have a superiority for many characters, compared to the average of their two parents. This superiority can be illustrated, for example, by better vigor, better yield, greater adaptation to the environment in which the hybrid is grown, and great uniformity of the hybrids compared to its parents. This hybrid vigor is all the more important as the parents are genetically distant.
  • the creation of pure lines involves the self-fertilization of a plant so as to obtain plants with the same genetic heritage, fixed for all the characteristics of productivity, regularity of yield, or resistance to the diseases, sought.
  • a first technique implemented, for obtaining pure lines consists in carrying out a chemical treatment of the plants so as to obtain plants capable of self-fertilization, for example hermaphrodite plants.
  • melon cucumis melo
  • the spraying of ethylene synthesis inhibitors such as silver nitrate or silver thiosulfate leads to the temporary appearance of stamens in female flowers (Rudich and al. , 1969; Risser et al. , 1979).
  • the transformation of gynoic plants into hermaphrodite plants is used for the maintenance of pure lines.
  • a first technique, implemented in particular for corn, consists in using mechanical means to carry out emasculation of the plants.
  • this technique proves to be extremely expensive since it requires the emasculation of each plant whose self-pollination is to be avoided, for each crossing carried out.
  • Another technique consists in carrying out a chemical emasculation of the plants, blocking the formation of viable pollen.
  • melons Cucumis melo
  • ethel precursor of ethylene
  • Another way to obtain plants capable of self-pollination useful for the creation of pure lines, or not capable of self-pollination, for the creation of hybrids could consist respectively of a selection of exclusively hermaphrodite, or exclusively female, individuals present in a species.
  • such a technique would also prove to be extremely expensive, since it would require the cultivation of a very large number of plants, until it is possible to determine the sexual type.
  • This technique would also be random, since the mechanisms for determining the sex of flowers depend in particular on environmental factors.
  • the PCT international application published under the number WO2007 / 125264 describes the identification and characterization of the genetic determinant (a), which has been found to consist of a gene encoding an aminocyclopropane carboxylate synthase (ACS). So the demand PCT No. WO 2007/125264 provides the detection and control means making it possible to select or generate dicotyledonous plants having the dominant allele (A) or the recessive allele (a).
  • the genetic determinant (g) remained completely unknown. At most, preliminary data suggested that the genetic determinant (g), of unknown nature and structure, could be located in a large genomic region of more than 2.4 megabases delimited by markers designated M8 and M30. Since it is commonly accepted that there are on average 12 open reading frames ("ORFs") in 100 kilobases of plant genome, the genomic region delimited between the markers M8 and M30 was likely to contain approximately 300 open frames of reading.
  • ORFs open reading frames
  • the genetic determinant (g) gynoic has been identified and characterized for the control of the floral development of a dicotyledonous plant which is an angiosperm, and more precisely a plant of the cucurbitaceae family .
  • the identification and characterization of the gynoic genetic determinant (g) made it possible for the first time to develop a combination of the two genetic determinants (a) andromonoic and (g) gynoic to fully control the development of the floral type. of a dicotyledonous plant, regardless of the sexual phenotype that is considered.
  • the invention therefore relates to a combination of the two genetic elements (A / a) and (G / g) which makes it possible to control the development of the floral type of a dicotyledonous plant, in particular of a cucurbitacea such as melon.
  • the inventors have now shown that, physiologically, the two alleles (G) and (g), which have been identified and characterized according to the invention, are distinguished by different levels of a new protein, the protein CmWIP1.
  • the application describes an ACS protein including the protein of sequence SEQ ID No. 3 or a protein having at least 90% amino acid identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% of amino acid identity, with the protein of sequence SEQ ID N ° 3.
  • the application describes a CmWIP1 protein including the protein of sequence SEQ ID No. 12 or a protein having at least 90% amino acid identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% of amino acid identity, with the protein of sequence SEQ ID No 12.
  • the protein CmWIP1 can also include the protein of sequence SEQ ID No 16 or a protein having at least 90 % identity in amino acids, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in amino acids, with the protein of sequence SEQ ID # 16.
  • the application also describes the regulatory polynucleotides (PG) and (Pg) as such
  • the invention also relates to methods for obtaining a transformed plant whose sexual phenotype has been modified, as well as the parts of such a plant, in particular its seeds.
  • a further subject of the invention is the protein CmWIP1 as defined in more detail below, or a fragment of this protein, as well as antibodies directed against the protein CmWIP1.
  • the invention also relates to methods for detecting the presence of alleles (A), (a), (G), and (g) in a sample.
  • the genetic determinant (G / g) which controls, in combination with the genetic determinant (A / a) previously described in the prior art, the development of the floral type in cucurbits has been identified and characterized. .
  • the identification and characterization of the genetic determinant provides for the first time to those skilled in the art the possibility of selecting or generating dicotyledonous plants having the desired sexual phenotype, in particular cucurbits such as the melon having the desired sexual phenotype.
  • the allele (A) controls the andromonoic character of the plants
  • the allele (G) controls the gynoic character of the plants, as illustrated in table 1 below.
  • Table 1 Phenotype genotype Type of flowers Monoecious AG- Males and females Andromonoic aaG- Males and hermaphrodites Hermaphrodite aagg Hermaphrodites Gynoic A-gg Females
  • Table 1 illustrates the correspondence between the genotype and the sexual phenotype of flowers of dicotyledonous plants.
  • the inventors have now shown that, physiologically, the two alleles (G) and (g), which have been identified and characterized according to the invention, are distinguished by different levels of a new protein, the protein CmWIP1.
  • the new protein CmWIP1 can be classified in the family of zinc finger proteins of the WIP type which are found in a wide variety of plants, including dicots, monocots, gymnosperms and mosses.
  • the inventors have shown that the allele (g) is distinguished from the allele (G) by a low level of protein CmWIP1 in the plant, compared to that of a plant carrying an allele (G). or by the production of a mutated CmWIP1 protein compared to the CmWIP1 protein produced by a plant carrying an allele (G).
  • the inventors have also shown that the allele (G) is dominant over the allele (g).
  • the two alleles (A) and (a) are distinguished by different rates of the enzymatic activity of a protein, the aminocyclopropane carboxylate synthase also designated ACS, which is a protein involved in the synthesis of ethylene.
  • allele (a) is physiologically distinguished from allele (A) by a low rate of enzymatic activity of ACS protein in the plant, compared to that of a plant carrying an allele (A ).
  • allele (A) is expressed in the carpel promordia and it is this expression which blocks the development of the stamens.
  • the absence of expression of allele A or the expression of a mutated form of the protein resulting from TILLING screens for example or of the protein resulting from allele a carrying the mutation A57V does not block the development of stamens .
  • allele (A) is dominant over allele (a).
  • the non-functional nucleic acid (NA) which characterizes the allele (a) includes (i) a nucleic acid coding for a protein distinct from the protein of SEQ ID No. 3, including the mutated protein A57V, (ii) or any other form of protein mutated with respect to the protein of sequence SEQ ID No. 3 and leading to an inactive enzyme, (ii) or other non-functional ACS (iv) or an unexpressed allele.
  • allele is meant within the meaning of the present invention, one of the forms of a gene occupying a site or locus on a pair of homologous chromosomes.
  • the alleles of a gene relate to the same genetic trait but can determine different phenotypes.
  • a dominant allele is an allele whose phenotypic expression level is much higher than that of the homologous allele (known as recessive). Dominance can be complete or partial.
  • a recessive allele is an allele that expresses itself in the phenotype only when the plant receives identical alleles from each of its two parents. On the other hand, the expression of the recessive allele is masked if the dominant homologous allele is present.
  • the plant has a monoecious or gynoic phenotype.
  • the plant When the first genetic control element (A / a) present in a plant is in the form of an allele (aa), the plant is of hermaphrodite or andromonoic phenotype.
  • the plant is of monoecious or andromonoic phenotype.
  • the plant is of hermaphrodite or gynoic phenotype.
  • Control genetic element G / g in the form of a dominant allele (G), in the combination of two genetic elements according to the invention.
  • the genetic control element G / g present in a plant in the form of the dominant allele (G), makes it possible to obtain a higher level of protein CmWIP1, compared to the level observed when the allele (G) is not present in said plant.
  • a "high level” of CmWIP1 protein corresponds to the average level of the CmWIP1 protein measured in a plant comprising the dominant allele (G) in its genome
  • a "low level »Of protein CmWIP1 corresponds to the average level of protein CmWIP1 observed in a plant not comprising the dominant allele (G) in its genome.
  • a functional regulator or promoter (PG) polynucleotide according to the invention consists of a nucleic acid which allows the expression of the protein CmWIP1 of sequence SEQ ID No. 12 in dicotyledonous plants.
  • such a promoter comprises, or consists of, the nucleic acid of sequence SEQ ID No. 13, which is located from the nucleotide in position 1 to the nucleotide in position 2999 of the nucleic acid of the CmWIP1 gene of sequence SEQ ID N ° 10.
  • the regulatory polynucleotide (PG) can consist of a polynucleotide which comprises, or which consists of, (i) a nucleotide sequence ranging from nucleotide 1 to nucleotide 2999 of the sequence SEQ ID No. 10 or (ii) a sequence having at least 90% nucleotide identity with the sequence 1-2999 of SEQ ID No. 10 and which is functional or (iii) a fragment of the preceding sequences (i) and (ii) and which is functional.
  • a subject of the invention is also the regulatory polynucleotide (PG) as such, defined above, as well as fragments of this nucleic acid, as will be described in more detail in the section entitled “Nucleic acids according to the invention.”
  • PG regulatory polynucleotide
  • a functional regulatory polynucleotide (PG) according to the invention may also consist of a promoter known to direct the expression of the nucleic acid sequence coding for the protein CmWIP1 in a constitutive manner or in a tissue-specific manner.
  • a functional regulatory polynucleotide (PG) according to the invention can thus be chosen from specific tissue promoters such as those of the genes of the “MADS box” family of the class ABCD and E, as described by Thei ⁇ en et al., 2001 or any other promoter of homeotic genes.
  • a functional regulatory polynucleotide (PG) according to the invention can also consist of an inducible promoter.
  • the subject of the invention is a combination of two genetic elements for the control of the floral type of a dicotyledonous plant, as defined above, in which the regulatory polynucleotide (PG) is sensitive to the action of an inducing signal. , and preferably, in which the regulatory polynucleotide (PG) is a polynucleotide activator inducible for transcription or translation.
  • the regulating polynucleotide activating transcription or translation is sensitive, directly or indirectly, to the action of an activating inducing signal, it is a polynucleotide "inducible activator" within the meaning of the invention.
  • a regulatory polynucleotide of the “inducible activator” type is a regulatory sequence which is only activated in the presence of an external signal.
  • an external signal can be the binding of a transcription factor, the binding of a transcription factor being able to be induced under the effect of the activating inducing signal to which the regulatory polynucleotide is directly or indirectly sensitive.
  • the expression of the polynucleotide coding for the protein CmWIP1 according to the invention can be induced by bringing the transformed cellular host into contact with the activating inducing signal to which the polynucleotide activator regulator is directly or indirectly sensitive.
  • the nucleic acid encoding the protein CmWIP1 comprises the nucleotide sequence SEQ ID No. 11.
  • the genetic control element (G / g) in the form of the recessive allele (g), when it is present in a plant which does not have the dominant allele (G) in its genome, does not make it possible to obtain a level of protein CmWIP1, as high as that obtained when the allele (G) is present.
  • the allele (g) as any alteration of the genotype corresponding to the allele (G), not making it possible to obtain a level of CmWIP1 as high as the allele (G).
  • a control element G / g in the form of a recessive allele is illustrated by the nucleic acid of sequence SEQ ID No. 15, which is an allele of the sequence coding for CmWIP1 in which is present a transposon nucleic acid designated “Gyno-hAT”.
  • the Gyno-hAT transposon is located from the nucleotide at position 7167 to the nucleotide at position 15412 of the sequence SEQ ID No. 15.
  • the Gyno-hAT transposon is located from the nucleotide in position 10 to the nucleotide in position 8246 of the sequence SEQ ID No. 14.
  • said polynucleotide (Pg) is methylated.
  • the regulatory polynucleotide (Pg) may consist of a regulatory polynucleotide (PG) which is in the form of a methylated nucleic acid.
  • methylated nucleic acid or "methylated regulatory polynucleotide” is meant according to the invention the corresponding nucleic acid whose ratio (number of methylated bases) / (number of nonmethylated bases) is at least 5/1.
  • a methylated nucleic acid includes a nucleic acid having a ratio (number of methylated bases) / (number of unmethylated bases) of at least 6/1, 7/1, 8/1, 9 / 1, 10/1, 11/1, 12/1, 13/1, 14/1, 15/1, 16/1, 17/1, 18/1 and 20/1.
  • the determination of the ratio (number of methylated bases) / (number of non-methylated bases) can be easily carried out by a person skilled in the art by any known technique. Those skilled in the art can in particular use the bisulfite sequencing method, as described in the examples of the present description.
  • a simple technique known to those skilled in the art consists in placing a selection marker gene under the control of the promoters to be tested.
  • a selection marker gene may for example be the gene for resistance to the herbicide BASTA, well known to those skilled in the art.
  • Another technique may consist in measuring the level of the protein CmWIP1 obtained when the sequence coding for this protein is under the control of different promoters, by using antibodies directed against this protein, and the methods described in the “polypeptides” part. according to the invention ".
  • an illustration of a non-functional promoter consists of a promoter having a nucleotide sequence identical to the nucleotide sequence of a functional promoter (PG) but which occurs in cellulo or in vivo under a non-functional methylated form.
  • the methylated state of the promoter (Pg) is caused by the presence of a transposable element (TE) located at a distance of less than 1 kilobases from the 3 'end of the CmWIP1 gene .
  • TE transposable element
  • a non-functional polynucleotide (Pg) can also be any polynucleotide derived from the polynucleotide (PG) as defined above, the nucleotide sequence of which includes an insertion, a substitution or a deletion of one or more nucleotides, with respect to the nucleotide sequence of the regulatory polynucleotide.
  • the subject of the invention is also a nucleic acid comprising a nucleotide sequence carrying at least one alteration chosen from a mutation, an insertion or a deletion, relative to the nucleic acid ranging from nucleotide 1 to nucleotide 2999 of the sequence SEQ ID No. 10, said altered nucleic acid leading to reduced expression of the protein CmWIP1, when it controls the expression of said protein, compared to the expression of the protein CmWIP1 controlled by the nucleic acid ranging from nucleotide 1 at nucleotide 2999 of the sequence SEQ ID No. 10.
  • Another subject of the invention is the regulatory polynucleotide (Pg) as such, as defined above.
  • the subject of the invention is also a nucleic acid comprising a regulatory polynucleotide (Pg) and a nucleic acid coding for the protein CmWIP1 of sequence SEQ ID No. 12.
  • Pg regulatory polynucleotide
  • the invention also relates to a combination of two genetic elements for controlling the development of the floral type of a dicotyledonous plant as defined generally in the present description, in which the regulatory polynucleotide (Pg) is sensitive to action of an inducing signal, and preferably, in which the regulatory polynucleotide (Pg) is a repressible polynucleotide inducible of transcription or translation.
  • repressor regulatory polynucleotide is meant, according to the invention, a regulatory sequence whose constitutive activity can be blocked by an external signal.
  • an external signal may be the absence of binding of a transcription factor recognized by the repressor regulatory polynucleotide. The absence of binding of the transcription factor can be induced under the effect of the repressor inducing signal to which the repressor regulatory polynucleotide is sensitive.
  • the expression of the sequence coding for a protein CmWIP1 is constitutive in the chosen cellular host, in the absence of the repressor inducing signal to which the repressor regulatory polynucleotide is directly or indirectly sensitive.
  • Contacting the cell host with the repressor inducing signal has the effect, by means of a direct or indirect action on the repressor regulatory polynucleotide, of inhibiting and / or blocking the expression of the polynucleotide coding for the protein CmWIP1.
  • Ng non-functional nucleic acid
  • a nucleic acid (Ng) includes nucleic acids comprising at least part of a sequence coding for an active CmWIP1 protein but which, when placed under the control of a functional regulatory polynucleotide, do not allow it to function in dicotyledonous plant cells , the production in said plants of an active CmWIP1 protein.
  • a nucleic acid (Ng) essentially includes nucleic acids (NG) in which one or more mutations are present in at least one intron or exon, each mutation being chosen from (i) the substitution of a nucleotide or more than one nucleotide, (ii) deletion of a nucleotide or at least two consecutive nucleotides and (ii) deletion of a nucleotide or at least two consecutive nucleotides, relative to the reference nucleic acid (NG) .
  • a nucleic acid (Ng) notably includes the nucleic acids coding for a non-active CmWIP1 protein.
  • nucleic acid coding for a non-active CmWIP1 protein is meant within the meaning of the present invention, a nucleic acid which codes for a protein which differs from the CmWIP1 protein of sequence SEQ ID No. 12, by substitution, deletion, or the insertion of one or more amino acids, and which does not possess the biological activity of the protein CmWIP1 of sequence SEQ ID No. 12.
  • nucleic acid which codes for a protein which differs from the protein CmWIP1 of sequence SEQ ID No. 16, by substitution, deletion, or insertion of one or more amino acids, and which does not have the biological activity of the protein CmWIP1 of sequence SEQ ID No. 16.
  • plants possessing the allele (g) of the genetic element (G / g) are obtained with nucleic acids encoding an inactive CmWIP1 protein.
  • plants possessing the allele (g) of the genetic element (G / g) were obtained with nucleic acids coding for a protein CmWIP1 having a substitution of a single nucleotide with respect to the nucleotide sequence SEQ ID No. 11 encoding the protein CmWIP1 of sequence SEQ ID No. 12.
  • the genetic control element A / a in the form of a dominant allele (A) or in the form of a dominant allele (a) has already been described in French patent application no. FR 2 900 415 and in the request PCT No. WO 2007/125264 .
  • the genetic control element (A / a) is an important element in the combination of two genetic elements for controlling the development of the floral type according to the invention, its main characteristics are again described below. .
  • Control genetic element A / a in the form of a dominant allele (A) of the combination according to the invention
  • the genetic control element A / a present in a plant in the form of the dominant allele (A), makes it possible to obtain a higher level of active ACS protein, compared to the level observed when l the allele (A) is not present in said plant.
  • a "high level” of ACS protein corresponds to the average level of ACS protein measured in a plant comprising the dominant allele (A) in its genome
  • a "low level »Of ACS protein corresponds to the rate means of active ACS protein observed in a plant not comprising the dominant allele (A) in its genome.
  • a low level of the ACS protein includes a zero level of active ACS protein, for example in the case of an expression of non-active ACS, including the product of the allele a carrying the mutation A57V.
  • PA - functional regulatory polynucleotide
  • a functional regulator or promoter (PA) polynucleotide according to the invention consists of a nucleic acid which allows the expression of the ACS protein of sequence SEQ ID No. 3 in dicotyledonous plants.
  • such a promoter comprises a nucleotide sequence ranging from nucleotide 1 to nucleotide 5906 of the sequence SEQ ID No. 1.
  • the regulatory polynucleotide (PA) can consist of a polynucleotide which comprises, or which consists of, (i) a nucleotide sequence ranging from nucleotide 1 to nucleotide 5906 of the sequence SEQ ID No. 1 or (ii) a sequence having at least 90% nucleotide identity with the sequence 1-5906 of SEQ ID No. 1 and which is functional or (iii) a fragment of the preceding sequences (i) and (ii) and which is functional.
  • the regulatory polynucleotide comprises or consists of a nucleotide sequence ranging from nucleotide 1 to nucleotide 5906 of the sequence SEQ ID No. 1.
  • a functional regulatory polynucleotide (PA) which is included in a combination of two genetic elements according to the invention may also consist of a promoter known to direct the expression of the nucleic acid sequence coding for the ACS protein constitutively or specific fabric way.
  • a functional regulatory polynucleotide (PA) included in a combination of two genetic elements according to the invention can thus be chosen from any of the constitutive or specific tissue promoters described above for certain embodiments of the regulatory polynucleotide (PG).
  • a functional regulatory polynucleotide (PA) according to the invention can also consist of an inducible promoter.
  • the subject of the invention is a combination of two genetic elements as defined above, in which the regulatory polynucleotide (PA) is sensitive to the action of an inducing signal, and preferably, in which the regulatory polynucleotide (PA) is a inducible transcription or translation activating polynucleotide, which can be chosen from any one of the inducible activating polynucleotides described in certain embodiments of the regulatory polynucleotide (PG).
  • the expression of the polynucleotide coding for the ACS protein according to the invention can be induced by bringing the transformed cellular host into contact with the activating inducing signal at which the polynucleotide activator regulator is directly or indirectly sensitive.
  • Control genetic element A / a in the form of a recessive allele (a) of the combination according to the invention
  • the allele (a) as any alteration of the genotype corresponding to the allele (A), not making it possible to obtain an active ACS level.
  • said polynucleotide (Pa) is methylated.
  • the regulatory polynucleotide (Pa) may consist of a regulatory polynucleotide (PA) which is in the form of a methylated nucleic acid.
  • methylated nucleic acid or "methylated regulatory polynucleotide” is meant according to the invention the corresponding nucleic acid whose ratio (number of methylated bases) / (number of nonmethylated bases) is at least 5/1.
  • a methylated nucleic acid includes a nucleic acid having a ratio (number of methylated bases) / (number of unmethylated bases) of at least 6/1, 7/1, 8/1, 9 / 1, 10/1, 11/1, 12/1, 13/1, 14/1, 15/1, 16/1, 17/1, 18/1 and 20/1.
  • the determination of the ratio (number of methylated bases) / (number of non-methylated bases) can be easily carried out by a person skilled in the art by any known technique. Those skilled in the art can in particular use the bisulfite sequencing method, as described in the examples of the present description.
  • a simple technique known to those skilled in the art consists in placing a selection marker gene under the control of the promoters to be tested.
  • a selection marker gene may for example be the gene for resistance to the herbicide BASTA, well known to those skilled in the art.
  • Another technique may consist in measuring the level of the ACS protein obtained when the sequence coding for this protein is under the control of different promoters, using antibodies directed against this protein, and the methods described in the “polypeptides” section. according to the invention ".
  • a non-functional regulatory polynucleotide comprises a nucleotide sequence ranging from nucleotide 1 to nucleotide 3650 of the sequence SEQ ID No. 2.
  • a non-functional regulatory polynucleotide comprises certain nucleotide sequences comprising one or more substitutions, deletions or additions of bases, relative to the nucleotide sequence ranging from nucleotide 1 to nucleotide 3650 of the sequence SEQ ID No. 1.
  • a non-functional regulatory polynucleotide can comprise a nucleotide sequence ranging from nucleotide 1 to nucleotide 3650 of the sequence SEQ ID No. 2 altered by one of the methods of I skilled in the art
  • the invention also relates to a combination of two genetic elements for controlling the development of the floral type as defined in the present description, and in which the allele (a) is a nucleic acid comprising the sequence SEQ ID No. 2 .
  • a nucleic acid of sequence SEQ ID No. 2 comprises a regulatory polynucleotide (Pa) and a nucleic acid coding for the ACS protein of sequence SEQ ID No. 3.
  • a non-functional polynucleotide (Pa) can also be any polynucleotide derived from the polynucleotide (PA) as defined above, the nucleotide sequence of which comprises an insertion, a substitution or a deletion of one or more nucleotides, with respect to the nucleotide sequence of the regulatory polynucleotide.
  • PA polynucleotide
  • a polynucleotide consists of a nucleic acid comprising a nucleotide sequence carrying at least one alteration chosen from a mutation, an insertion or a deletion, with respect to the nucleic acid ranging from nucleotide 1 to nucleotide 5907 of the sequence SEQ ID No. 1, said altered nucleic acid leading to reduced expression of the ACS protein, when it controls the expression of said protein, compared to the expression of the ACS protein controlled by the nucleic acid ranging from nucleotide 1 to nucleotide 5907 of the sequence SEQ ID No. 1.
  • the invention also relates to a combination of two genetic elements for controlling the development of the floral type as defined above, in which the regulatory polynucleotide (Pa) is sensitive to the action of an inducing signal, and preferably, in which the regulatory polynucleotide (Pa) is a repressor-inducible polynucleotide of transcription or translation.
  • the expression of the sequence coding for an ACS protein is constitutive in the chosen cellular host, in the absence of the repressor inducing signal to which the repressor regulatory polynucleotide is directly or indirectly sensitive.
  • Contacting the cell host with the repressor inducing signal has the effect, by means of a direct or indirect action on the repressor regulatory polynucleotide, of inhibiting and / or blocking the expression of the polynucleotide encoding the ACS protein.
  • a nucleic acid includes nucleic acids comprising at least part of a sequence coding for an active ACS protein but which, when placed under the control of a functional regulatory polynucleotide, do not make it possible in dicotyledonous plant cells , the production in said plants of an active CmWIP1 protein.
  • a nucleic acid (Na) essentially includes nucleic acids (NA) in which one or more mutations are present in at least one intron or exon, each mutation being chosen from (i) the substitution of a nucleotide or more than one nucleotide, (ii) deletion of a nucleotide or at least two consecutive nucleotides and (ii) deletion of a nucleotide or at least two consecutive nucleotides, relative to the reference nucleic acid (NA) .
  • a nucleic acid (Na) notably includes the nucleic acids coding for a non-active ACS protein.
  • nucleic acid coding for a non-active ACS protein means a nucleic acid which codes for a protein which differs from the ACS protein of sequence SEQ ID No. 3, by substitution, deletion, or the insertion of one or more amino acids, and which does not possess the biological activity of the ACS protein of sequence SEQ ID No. 3.
  • An illustrative example of such a nucleic acid is presented in the figure 8 .
  • such a non-active ACS protein does not make it possible to transform S-adenosyl methionine into ACC (1-aminocyclopropane-1-carboxylate).
  • allelic variants (G) and (g) of the second genetic control element (G / g) included in a combination of two genetic elements for controlling the floral development of the invention have been characterized.
  • the inventors identified the nucleic acid of sequence SEQ ID No. 10 as being a nucleic acid corresponding to the dominant allelic variant (G) and its methylated form in planta , as corresponding to the recessive allelic variant (g), of the second genetic element of control in the form of a gene (G / g).
  • At least the second of the two genetic control elements has been introduced artificially into a plant.
  • nucleic acid of sequence SEQ ID No. 10 is one of the objects of the invention.
  • the subject of the present invention is therefore a nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with the nucleotide sequence SEQ ID No. 10, or with a fragment of the sequence SEQ ID No. 10, provided that such a nucleic acid has the functional characteristics of the allele (G) as defined above.
  • nucleic acid of sequence complementary to the nucleic acid as defined above.
  • nucleic acid consisting of a polynucleotide having at least 95% nucleotide identity with the sequence SEQ ID No. 10, or with a fragment of the sequence SEQ ID No. 10, or an acid nucleic acid of complementary sequence, provided that such a nucleic acid has the functional characteristics of the allele (G) as defined above.
  • the invention also relates to a nucleic acid comprising at least 12, preferably at least 15 and very preferably at least 20 consecutive nucleotides of the nucleic acid of sequence SEQ ID No. 10, it being understood that such nucleic acid includes in its definition the "fragments" of a nucleic acid according to the invention as defined in the present description.
  • the invention also relates to the nucleic acid comprising or consisting of the sequence SEQ ID No. 10.
  • the invention also relates to a nucleic acid comprising at least 12, preferably at least 15 and very preferably at least 20 consecutive nucleotides of the nucleic acid of sequence SEQ ID No. 10, it being understood that such nucleic acid includes in its definition the "fragments" of a nucleic acid according to the invention as defined in the present description.
  • the invention also relates to a nucleic acid comprising at least 12 consecutive nucleotides of an exonic polynucleotide of the G / g gene, such as the polynucleotides 1 and 2 described in table 2 above, which are included in the acid nucleic acid with sequence SEQ ID N ° 10
  • Such a nucleic acid codes for at least part of the protein CmWIP1 and can in particular be inserted into a recombinant vector intended for the expression of the corresponding translation product in a host cell or in a plant transformed with this recombinant vector, with a view to to obtain a plant of genotype (G).
  • Such a nucleic acid can also be used for the synthesis of nucleotide probes and primers intended for the detection or the amplification of nucleotide sequences included in the gene (G / g) in a sample.
  • sequences described above can carry one or more mutations, preferably one or more mutations such as to induce the synthesis of a non-active CmWIP1 protein, and to modify the sexual type of a plant carrying such a gene ( G / g) mutated.
  • Such sequences meet the definition of nucleic acids coding for a non-active CmWIP1 protein, generally defined above.
  • the invention also relates to a nucleic acid comprising at least 12 consecutive nucleotides of an intronic polynucleotide of the gene (G / g), described in table 3 above, which are included in the nucleic acid of sequence SEQ ID # 10.
  • Such a nucleic acid can be used as an oligonucleotide probe or primer to detect the presence of at least one copy of the gene (G / g) in a sample, or even to amplify a determined target sequence within the gene (A / a) .
  • Such a nucleic acid can also be used to amplify a specific target sequence within the gene (G / g) or to inhibit it by a sense or co-suppression approach, or by the use of double-stranded RNA (Wassenegger et al 1996; Kooter et al. 1999) for interference.
  • Such a nucleic acid can also be used to search for functional allelic variants of the gene (G / g), which can be used in a method of selecting plants having a determined sexual type.
  • nucleic acids according to the invention coding for the protein CmWIP1
  • the subject of the invention is also a nucleic acid comprising a polynucleotide having at least 95% identity in nucleotides with the nucleotide sequence starting at the nucleotide in position 3000 and ending at the nucleotide in position 5901 of the sequence SEQ ID No. 10 thus than a nucleic acid of complementary sequence.
  • the invention also relates to a nucleic acid having at least 95% nucleotide identity with the nucleotide sequence starting at the nucleotide at position 3000 and ending at the nucleotide at position 5901 of the sequence SEQ ID No. 10, as well as an acid nucleic acid of complementary sequence.
  • the subject of the invention is also a nucleic acid comprising the nucleotide sequence starting at the nucleotide in position 3000 and ending at the nucleotide in position 5901 of the sequence SEQ ID No. 10 or a nucleic acid of complementary sequence.
  • the invention also relates to a nucleic acid consisting of the nucleotide sequence starting at the nucleotide at position 3000 and ending at the nucleotide at position 5901 of the sequence SEQ ID No. 10 or a nucleic acid of complementary sequence.
  • a nucleic acid coding for the protein CmWIP1 can further comprise leader and terminator sequences, conventional for those skilled in the art.
  • the expression of the genomic nucleic acid encoding the protein CmWIP1 leads to the synthesis of a messenger RNA whose cDNA is the nucleic acid of sequence SEQ ID No. 11, which is also one of the objects of the present invention.
  • a further subject of the invention is therefore the polypeptide comprising the amino acid sequence SEQ ID No. 12, also called “protein CmWIP1” in the present description, as well as a polypeptide having at least 95% identity in amino acids with the sequence SEQ ID No. 12, or a fragment or a variant thereof.
  • An illustration according to the invention of a protein CmWIP1 having at least 95% amino acid identity with the sequence SEQ ID No. 12 consists of the protein of sequence SEQ ID No. 16, which differs from the protein of sequence SEQ ID No. 12 by deleting a Serine residue.
  • a fragment of a CmWIP1 protein according to the invention comprises at least 10, 50, 100, 200, 300, 320, 330, 340, 345 or 353 consecutive amino acids of a polypeptide of sequence SEQ ID No. 12.
  • the invention also relates to a polypeptide comprising an amino acid sequence having at least 95% amino acid identity with the sequence of a CmWIP1 protein of sequence SEQ ID No. 12.
  • polypeptide having at least 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5% , 99.6%, 99.7%, 99.8% or 99.9% of amino acid identity with the sequence of a polypeptide of sequence SEQ ID No. 12, or a peptide fragment of the latter.
  • polypeptides according to the present invention are in an isolated or purified form.
  • a polypeptide according to the invention can be obtained by genetic recombination according to techniques well known to those skilled in the art, for example techniques described in AUSUBEL et al. (1989).
  • a polypeptide according to the invention can also be prepared by conventional techniques of chemical synthesis, either in homogeneous solution or in solid phase.
  • a polypeptide according to the invention may be prepared by the technique in homogeneous solution described by HOUBEN WEIL (1974) or also by the solid phase synthesis technique described by MERRIFIELD (1965a; 1965b).
  • variant polypeptides of a polypeptide according to the invention retain their ability to be recognized by antibodies directed against the polypeptides of sequences SEQ ID No. 12.
  • a polypeptide encoded by the gene (G / g) according to the invention such as a polypeptide of amino acid sequence SEQ ID No. 12, or a variant or a peptide fragment of the latter is useful in particular for the preparation of antibody intended for the detection of the presence and / or the expression of a polypeptide of sequences SEQ ID No. 12 or of a peptide fragment of the latter in a sample.
  • antibodies directed against these polypeptides are used to quantify the synthesis of a polypeptide of sequences SEQ ID No. 12, for example in cells of a plant, and thus determine the sex of the plant, without having to cultivate it.
  • antibody within the meaning of the present invention, is meant in particular polyclonal or monoclonal antibodies or fragments (for example the fragments F (ab) ' 2 , F (ab)) or any polypeptide comprising a domain of the antibody initial recognizing the polypeptide or the fragment of target polypeptide according to the invention.
  • Monoclonal antibodies can be prepared from hybridomas using the technique described by KOHLER and MILSTEIN (1975)
  • the present invention also relates to antibodies directed against a polypeptide as described above or a fragment or a variant thereof, as produced in the trioma technique or also the hybridoma technique described by KOZBOR et al. (1983).
  • the invention also relates to fragments of single chain Fv antibody (ScFv) as described in the patent. US No. 4,946,778 or by MARTINEAU et al. (1998).
  • the antibodies according to the invention also comprise fragments of antibodies obtained using phage libraries as described by RIDDER et al. (1995) or humanized antibodies as described by REINMANN et al. (1997) and LEGER et al. (1997).
  • the antibody preparations according to the invention are useful in immunological detection tests intended to identify the presence and / or the amount of a polypeptide SEQ ID NO: 3, or a peptide fragment thereof, present in a sample.
  • An antibody according to the invention may also comprise an isotopic or non-isotopic detectable marker, for example fluorescent, or else be coupled to a molecule such as biotin, according to techniques well known to those skilled in the art.
  • an isotopic or non-isotopic detectable marker for example fluorescent
  • a molecule such as biotin
  • Another subject of the invention consists in the use of a nucleic acid or an allelic variant of a nucleic acid as defined above in plant selection programs for obtaining plants of which the type floral has been changed.
  • Nucleic acids comprising a functional regulatory polynucleotide (PG)
  • a functional regulator or promoter (PG) polynucleotide according to the invention consists of a nucleic acid which allows the expression of the protein CmWIP1 of sequence SEQ ID No. 12 in dicotyledonous plants.
  • Such a functional regulatory polynucleotide (PG) thus makes it possible, when it is artificially introduced into a plant, to modify the sex of the flowers of such a plant, and in particular makes it possible to obtain male and female or male and hermaphrodite plants, capable of self-pollination
  • the invention therefore also relates to a nucleic acid comprising a polynucleotide having at least 95% identity in nucleotides with the nucleotide sequence starting at the nucleotide in position 1 and ending at the nucleotide in position 2999 of the sequence SEQ ID N ° 10 as well as a nucleic acid of complementary sequence.
  • the invention also relates to a nucleic acid having at least 95% nucleotide identity with the nucleotide sequence starting at the nucleotide in position 1 and ending at the nucleotide in position 2999 of the sequence SEQ ID No. 10, as well as an acid nucleic acid of complementary sequence.
  • the subject of the invention is also a nucleic acid comprising the nucleotide sequence starting at the nucleotide in position 1 and ending at the nucleotide in position 2999 of the sequence SEQ ID No. 10 or a nucleic acid of complementary sequence.
  • the invention also relates to a nucleic acid consisting of the nucleotide sequence starting at the nucleotide in position 1 and ending at the nucleotide in position 2999 of the sequence SEQ ID No. 10 or a nucleic acid of complementary sequence.
  • the regulatory polynucleotide ranging from the nucleotide in position 1 to the nucleotide in position 2999 of the sequence SEQ ID No. 10 is also referenced as the nucleic acid of sequence SEQ ID No. 13 in the present description.
  • the invention also relates to a nucleic acid comprising at least 12 consecutive nucleotides of a regulatory polynucleotide, as defined above.
  • Such a nucleic acid can be used as a probe or oligonucleotide primer to detect the presence of at least one copy of the allele (G) of the gene (G / g) in a sample, to amplify a determined target sequence within the gene (G / g).
  • Such a nucleic acid can also be used to search for functional allelic variants of the gene (G / g), or may be used in a method of selecting plants having a determined sexual type.
  • Such a nucleic acid can also be used to inhibit a determined target sequence within the gene (G / g) by an antisense or co-suppression approach, or by the use of double-stranded RNA (Wassenegger et al. 1996; Kooter et al. 1999) for interference.
  • Nucleic acids comprising a non-functional Pg regulatory polynucleotide
  • Such a non-functional regulatory polynucleotide (Pg) thus makes it possible, when it is introduced artificially into a plant, for example in replacement of a polynucleotide (G), to modify the sex of the flowers of such a plant, and in particular allows d '' obtain hermaphrodite plants, capable of self-pollination, or female plants.
  • Such a nucleic acid can be used as a probe or oligonucleotide primer to detect the presence of at least one copy of the allele (a) of the gene (G / g) in a sample, or also to amplify a target sequence determined within of the gene (G / g).
  • nucleic acids comprising the association of one or more nucleic acids as defined above, for example a nucleic acid coding for a functional protein CmWIP1 under the control of a promoter of type (PG) or (Pg ).
  • PG promoter of type
  • Pg promoter of type
  • any conventional technique of molecular biology, microbiology and recombinant DNA known to those skilled in the art can be used. Such techniques are described for example by SAMBROOK et al. (1989), GLOVER (1985), GAIT (1984), HAMES and HIGGINS (1984), BERBAL (1984) and AUSUBEL et al. (1994).
  • any nucleic acid and any polypeptide according to the invention is in an isolated or purified form.
  • isolated in the sense of the present invention designates a biological material which has been removed from its original environment (the environment in which it is naturally located).
  • a polynucleotide found naturally in a plant is not isolated.
  • the same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted into the genome of the plant is isolated.
  • Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment.
  • purified does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
  • a polynucleotide or a polypeptide is in the purified state after purification of the starting material or of the natural material of at least one order of magnitude, preferably 2 or 3 and preferably four or five orders of magnitude.
  • nucleotide sequence can be used to denote either a polynucleotide or a nucleic acid.
  • nucleotide sequence encompasses the genetic material itself and is therefore not limited to information regarding its sequence.
  • nucleic acid include RNA, DNA, cDNA sequences or even RNA / DNA hybrid sequences of more than one nucleotide, in single strand form or in duplex form.
  • nucleotide designates both natural nucleotides (A, T, G, C) as well as modified nucleotides which comprise at least one modification such as (i) an analog of a purine, (ii) an analog of d 'a pyrimidine, or (iii) a similar sugar, such modified nucleotides being described for example in the application PCT No. WO 95/04064 .
  • a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired with the base complementary to the second polynucleotide whose orientation is reversed.
  • the complementary bases are A and T (or A and U), and C and G.
  • a first nucleic acid having at least 95% identity with a second reference nucleic acid will have at least 95%, preferably at least 96%, 97%, 98%, 98.5%, 99 %, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity nucleotides with this second reference polynucleotide, the percentage of identity between two sequences being determined as described below.
  • the “percentage identity” between two nucleic acid sequences is determined by comparing the two optimally aligned sequences, through a comparison window.
  • the part of the nucleotide sequence in the comparison window can thus include additions or deletions (for example “gaps”) with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain a optimal alignment between the two sequences.
  • additions or deletions for example “gaps”
  • the percentage of identity is calculated by determining the number of positions at which an identical nucleic base is observed for the two sequences compared, then by dividing the number of positions at which there is identity between the two nucleic bases by the total number of positions in the comparison window, then multiplying the result by one hundred in order to obtain the percentage of nucleotide identity of the two sequences between them.
  • the optimal alignment of the sequences for the comparison can be carried out by computer using known algorithms.
  • a nucleic acid having at least 95% nucleotide identity with a nucleic acid according to the invention includes the "variants" of a nucleic acid according to the invention.
  • variant of a nucleic acid according to the invention is meant a nucleic acid which differs from the reference nucleic acid by one or more substitutions, additions or deletions of a nucleotide, relative to the nucleic acid of reference.
  • a variant of a nucleic acid according to the invention can be of natural origin, such as an allelic variant which exists naturally. Such a variant nucleic acid can also be an unnatural nucleic acid obtained, for example, by mutagenesis techniques.
  • the differences between the reference nucleic acid and the "variant" nucleic acid are reduced so that the reference nucleic acid and the variant nucleic acid have very similar nucleotide sequences and, in many regions , identical.
  • the nucleotide modifications present in a variant nucleic acid can be silent, which means that they do not affect the amino acid sequence which can be encoded by this variant nucleic acid.
  • Changes in nucleotides in the variant nucleic acid can also result in substitutions, additions or deletions of one or more amino acids in the sequence of the polypeptide which can be encoded by this variant nucleic acid.
  • a variant nucleic acid according to the invention comprising an open reading phase, code for a polypeptide which retains the same function or the same biological activity as the polypeptide coded by the reference nucleic acid.
  • a variant nucleic acid according to the invention and which comprises an open reading phase codes for a polypeptide which retains the capacity to be recognized by antibodies directed against the polypeptide encoded by the nucleic acid of reference.
  • fragment of a nucleic acid according to the invention is meant a nucleotide sequence of a reduced length compared to the reference nucleic acid, the nucleic acid fragment having a nucleotide sequence identical to the nucleotide sequence of the reference nucleic acid on the common part.
  • Such fragments of a nucleic acid according to the invention have at least 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300 , 400, 500, 1000, 2000 or 3000 consecutive nucleotides of the reference nucleic acid, the maximum length in nucleotides of a fragment of a nucleic acid according to the invention is of course limited by the maximum length in nucleotides of l reference nucleic acid.
  • nucleic acids according to the invention are useful for detecting the presence of at least one copy of a nucleotide sequence of the gene (G / g) or a fragment or an allelic variant of the latter in a sample.
  • the above probes and primers derived from the sequence SEQ ID No. 10, and in particular derived from the regulatory polynucleotide (PG) can be used to detect the presence of the allele (G) in a dicotyledonous plant.
  • nucleotide probes and primers hybridizing, under high stringency hybridization conditions, with a nucleic acid chosen from the sequences SEQ ID No. 10 and SEQ ID No. 11, or with a regulatory polynucleotide ( PG) or (Pg).
  • PG regulatory polynucleotide
  • the invention therefore also relates to a nucleic acid, usable as a probe or primer, specifically hybridizing with a nucleic acid as defined above.
  • hybridization conditions below are implemented for the hybridization of a nucleic acid, probe or primer, 20 bases in length.
  • the parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
  • Tm 81.5 + 0.41 % G + VS + 16.6 Log concentration in cations - 0.63 % formamide - 600 / number of basics SAMBROOK and al . , 1989 , pages 9.54 - 9.62 .
  • Tm 4 (G + C) + 2 (A + T).
  • the hybridization temperature is approximately 5 to 30 ° C, preferably 5 to 10 ° C below Tm.
  • high stringency hybridization conditions hybridization conditions such that one places oneself at a hybridization temperature of 5 ° C. below the Tm.
  • hybridization conditions described above can be adapted as a function of the length and of the base composition of the nucleic acid whose hybridization is sought or of the type of labeling chosen, according to techniques known to those skilled in the art. job.
  • the suitable hybridization conditions can for example be adapted according to the teaching contained in the work of HAMES and HIGGINS (1985) or also in the work of AUSUBEL et al. (1989).
  • hybridization conditions used for a nucleic acid 200 bases in length are as follows:
  • 5 x SSPE (0.9 M NaCl, 50 mM sodium phosphate pH 7.7, 5 mM EDTA)
  • 5 x Denhardt's (0.2% PVP, 0.2% Ficoll, 0.2% SAB) 100 ⁇ g / ml DNA of salmon sperm 0.1% SDS duration: 1 night.
  • the nucleotide probes or primers according to the invention comprise at least 12 consecutive nucleotides of a nucleic acid according to the invention, in particular of a nucleic acid of sequences SEQ ID No 10 or SEQ ID No 11 or its sequence complementary, of a nucleic acid having 95% identity in nucleotides with a sequence chosen from the sequences SEQ ID N ° 10 or SEQ ID N ° 11 or of its complementary sequence or of a nucleic acid hybridizing under conditions d of high stringency with a sequence chosen from the sequences SEQ ID No. 10 or SEQ ID No. 11 or its complementary sequence.
  • nucleotide probes or primers according to the invention will have a length of at least 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 100, 150, 200, 300, 400 , 500, 1000, 2000 or 3000 consecutive nucleotides of a nucleic acid according to the invention.
  • a probe or a nucleotide primer according to the invention will consist and / or include fragments with a length of 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 100, 150, 200, 300, 400, 500, 1000, 2000 or 3000 consecutive nucleotides of a nucleic acid according to the invention.
  • a primer or a nucleotide probe according to the invention can be prepared by any suitable method well known to those skilled in the art, including by cloning and action of restriction enzymes or also by direct chemical synthesis according to techniques such as the method to the phosphodiester of NARANG et al. (1979) or BROWN et al. (1979), the diethylphosphoramidite method of BEAUCAGE et al. (1980) or the solid support technique described in European patent no. EP 0 707 592 .
  • Each of the nucleic acids according to the invention can be labeled, if desired, by incorporating a detectable molecule, that is to say a detectable marker, by spectroscopic means, photochemical, biochemical, immunochemical or even chemical.
  • markers can consist of radioactive isotopes ( 32 P 3 H, 35 S), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetylaminofluorene) or also ligands such as biotin.
  • the labeling of the probes is preferably done by incorporating labeled molecules within the polynucleotides by extension of primers, or else by adding to the 5 ′ or 3 ′ ends.
  • the probes according to the invention can have structural characteristics such as to allow amplification of the signal, such as the probes described by URDEA et al; (1991) or in European patent no. EP 0 225 807 (Chiron ).
  • oligonucleotide probes according to the invention can be used in particular in Southern type hybridizations to any nucleic acid coding for the protein CmWIP1, in particular the nucleic acids of sequences SEQ ID No. 10 or SEQ ID No. 11, or also in hybridizations to RNA when the expression of the corresponding transcript is sought in a sample.
  • the probes according to the invention can also be used for the detection of PCR amplification products or even for the detection of mismatches.
  • Nucleotide probes or primers according to the invention can be immobilized on a solid support.
  • Such solid supports are well known to those skilled in the art and comprise surfaces of the wells of microtitration plates, polystyrene beads, magnetic beads, nitrocellulose strips or even microparticles such as latex particles.
  • the subject of the invention is also a nucleic acid which can be used as a nucleotide probe or primer, characterized in that it comprises at least 12 consecutive nucleotides of a nucleic acid as defined above, in particular of a nucleic acid with nucleotide sequences SEQ ID No. 10 and SEQ ID No. 11.
  • the invention also relates to a nucleic acid which can be used as a nucleotide probe or primer, characterized in that it consists of a polynucleotide of at least 12 consecutive nucleotides of a nucleic acid according to the invention, quite preferred of a nucleic acid of sequences chosen from the nucleotide sequences SEQ ID No. 10 and SEQ ID No. 11.
  • such a nucleic acid can further be characterized in that it is labeled with a detectable molecule.
  • nucleic acids, and the regulatory polynucleotides defined above must be introduced into vectors, then into cells.
  • the invention also relates to vectors, cells and transformed plants, which include regulatory polynucleotides (PG) and (Pg), nucleic acids coding for active or non-active CmWIP1 proteins, as well as nucleic acids. corresponding to alleles (G) and (g) as described above, and the primers defined above.
  • PG regulatory polynucleotides
  • Pg regulatory polynucleotides
  • G alleles
  • G alleles
  • a nucleic acid as defined above hereinafter called the nucleic acid of interest, can be inserted into an appropriate vector.
  • vector in the sense of the present invention, is meant a circular or linear DNA or RNA molecule which is either in single strand or double strand form.
  • a recombinant vector according to the invention is preferably an expression vector, or more specifically an insertion vector, a transformation vector or an integration vector.
  • It may especially be a vector of bacterial or viral origin.
  • the nucleic acid of interest is placed under the control of one or more sequences containing signals for regulating its expression in the plant in question, that is to say that the regulatory signals are all contained in the nucleic acid. of interest, as is the case in the nucleic acid constructs described in the previous section, either that one, several of them, or even all of the regulatory signals are contained in the recipient vector in which the nucleic acid of interest has been inserted.
  • a recombinant vector according to the invention advantageously comprises appropriate sequences for initiating and stopping transcription.
  • the recombinant vectors according to the invention can include one or more origins of functional replication in the host cells in which their expression is sought, as well as, where appropriate, nucleotide selection marker sequences.
  • the recombinant vectors according to the invention can also include one or more of the expression regulation signals as defined above in the description.
  • the preferred bacterial vectors according to the invention are, for example, the vectors pBR322 (ATCC No. 37,017) or else the vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, WI, United States) ).
  • They may also be vectors of the Baculovirus type such as the vector pVL1392 / 1393 (Pharmingen) used to transfect the cells of the line Sf9 (ATCC No. CRL 1711) derived from Spodoptera frugiperda.
  • vectors of the Baculovirus type such as the vector pVL1392 / 1393 (Pharmingen) used to transfect the cells of the line Sf9 (ATCC No. CRL 1711) derived from Spodoptera frugiperda.
  • the most common methods for introducing nucleic acids into bacterial cells can be used in the context of this invention. It can be the fusion of receptor cells with bacterial protoplasts containing DNA, electroporation, projectile bombardment, infection with viral vectors, etc.
  • Bacterial cells are often used to amplify the number of plasmids containing the construct comprising the nucleotide sequence, object of the invention.
  • the bacteria are cultured and the plasmids are then isolated according to methods well known to those skilled in the art (see the manuals of protocols already cited), including the plasmid purification kits sold commercially such as EasyPrepl from Pharmacia Biotech or QIAexpress Expression System from Qiagen.
  • the plasmids thus isolated and purified are then manipulated to produce other plasmids which will be used to transfect plant cells.
  • nucleic acids or the recombinant vectors defined in the present description must be introduced into a host cell.
  • the introduction of the polynucleotides according to the invention into a host cell can be carried out in vitro , according to techniques well known to those skilled in the art.
  • the invention further relates to a host cell transformed with a nucleic acid according to the invention or with a recombinant vector as defined above.
  • Such a transformed host cell is preferably of bacterial, fungal or vegetable origin.
  • bacterial cells of different strains of Escherichia coli or of Agrobacterium tumefaciens can be used .
  • the transformed host cell is a plant cell or even a plant protoplast.
  • it is a cell or a protoplast of a plant belonging to the species cucumis melo.
  • the subject of the invention is also the use of a nucleic acid of interest, for manufacturing a transformed plant whose sexual phenotype is modified.
  • the invention also relates to the use of a recombinant vector as defined in the present description for manufacturing a transformed plant whose sexual phenotype is modified.
  • the invention also relates to the use of a cellular host transformed with a nucleic acid of interest, for manufacturing a transformed plant whose sexual phenotype is modified.
  • the invention also relates to a transformed plant comprising a plurality of host cells as defined above.
  • the invention also relates to a transformed multicellular plant organism, characterized in that it comprises a transformed host cell or a plurality of host cells transformed by at least one of the nucleic acids as defined above, or also by a vector. recombinant comprising such a nucleic acid.
  • the transformed plant may contain a plurality of copies of a nucleic acid coding for the protein CmWIP1, in situations in which an overexpression of the protein CmWIP1 is sought.
  • An overexpression of the protein CmWIP1 is sought in particular when it is desired to obtain plants producing male and female flowers or else plants producing male and hermaphrodite flowers.
  • a plant overexpressing the protein CmWIP1 produces male and female flowers in the embodiments of the combination of two genetic elements of the invention in which the ACS protein is also overexpressed (allele A now in the homozygous or heterozygous state).
  • a plant overexpressing the protein CmWIP1 produces male and hermaphrodite flowers in the embodiments of the combination of two genetic elements of the invention in the absence of expression of an active ACS protein.
  • the invention therefore also relates to a transformed plant as defined above, the flowers of which are male and female, and to a transformed plant as defined above, the flowers of which are male and hermaphrodite.
  • the transformed plant may contain a plurality of copies of a nucleic acid encoding the ACS protein, in situations where overexpression of the ACS protein is wanted.
  • An overexpression of the ACS protein is sought in particular when it is desired to obtain plants producing female flowers, not capable of self-pollination.
  • a plant overexpressing the ACS protein produces male and female flowers in the embodiments of the combination of two genetic elements of the invention in which the protein CmWIP1 is also overexpressed (allele G present in the homozygous state).
  • a plant overexpressing the ACS protein produces female flowers in the embodiments of the combination of two genetic elements of the invention in the absence of expression of an active CmWIP1 protein.
  • the invention therefore also relates to a transformed plant as defined above whose flowers are male and female and to a transformed plant as defined above whose flowers are exclusively female.
  • the plants transformed according to the invention all comprise at least the second genetic element of the combination according to the invention, chosen from nucleic acids, and the regulatory polynucleotides defined above in a form artificially introduced into their genome.
  • the invention also relates to any part of a transformed plant as defined in the present description, such as the root, but also the aerial parts such as the stem, the leaf, the flower and especially the seed or the fruit.
  • the subject of the invention is also a seed or a seed of a plant produced by a transformed plant as defined above.
  • such a transformed seed or such a transformed grain comprises one or more cells comprising in their genome one or more copies of the first and second genetic control elements as defined above, artificially introduced into said dicotyledonous plant allowing the synthesis of protein CmWIP1 at a high or a low level, if necessary in a controlled and inducible manner.
  • a transformed plant it is sought to express in a controlled manner the protein CmWIP1, which implies that the transformed plant does not contain, as a functional copy of a polynucleotide encoding the protein CmWIPI, only the copy or copies which have been artificially introduced into their cells, and preferably into their genome, whereas the gene sequences (G / g) coding for CmWIP1, found naturally in the wild plant carry at least one mutation causing a defect in gene expression (G / g).
  • the plants transformed according to the invention are dicots, preferably belonging to the cucurbitaceae family , and in particular to the genera chosen from: Abobra, Acanthosicyos, Actinostemma, Alsomitra, Ampelosicyos, Anacaona, Apat3ingania, Apodanthera, Bambekea, Benincasa, Biswarea, Bolbostemma, Brandegea, bryonia, Calycophysum, Cayaponia, Cephalopentandra, Ceratosanthes, Chalema, Cionosicyos, Citrullus, Coccinia, Cogniauxia, Corallocarpus, Cremastopus, Ctenolepis, Cucumella, Cucumeropsis, Cucumis, Cucurbita ,, Cucurbitella, Dicantis Ditro, Dantro, Dantro, Dantro, Dantocell, , Ecballium, Echinocystis, Echinope
  • the transformed plants belong to the genus cucumis, and to the species Cucumis Melo.
  • the identification of the system for controlling the development of the floral type by the inventors has made it possible to develop extremely simple methods for detecting the sexual phenotype of plants, the main characteristics of which are detailed below.
  • the detection of the complex between a nucleic acid and a probe can be done by any technique known to those skilled in the art, and in particular by using labeled probes or primers, as described in the section "Probes and primers according to the invention "
  • TILLING for "Targeted Induced Local Lesions IN Genomes" is a reverse genetic method which is based on the ability of an endonuclease to detect mismatches in a double strand of DNA and to cut the DNA at the level of unpaired bases. This technique makes it possible to detect unique mutation points generated by the exposure of plants to a mutagenic chemical compound.
  • the TILLING technique thus allows the identification of a series of alleles of a given gene and is particularly well suited to the implementation of high throughput screening methods allowing the selection, in target genes of interest, mutations induced by chemical mutagenesis.
  • the Tilling technique is particularly well suited to obtaining and selecting plants into which the allele (g) of the second genetic control element (G / g) has been artificially introduced.
  • the Tilling technique can also be used successfully for obtaining and selecting plants into which the allele (a) of the first genetic element (A / a) has been artificially introduced.
  • the Tilling technique is suitable for obtaining and selecting plants in which we have artificially introduced both the allele (g) of the second genetic control element (G / g) and the allele (a) of the first genetic element (A / a).
  • step a) is carried out by chemical mutagenesis of the seeds of the dicotyledonous plants of interest, by exposure of the seeds to a mutagenic agent, for example to ethyl methanesulfonate, for example using the method described by Koornbeef et al. (1982, Mutat Res, Vol. 93: 109-123 ).
  • a mutagenic agent for example to ethyl methanesulfonate
  • M1 plants are generated from seeds previously exposed to the mutagen.
  • the M1 plants are then self-fertilized in order to generate a collection of M2 plants which is the collection of mutant plants generated in step a) of the process.
  • step b) the DNA of each plant is extracted from the collection of plants generated in step a) and an amplification of the nucleic acid of the target gene is carried out, here the gene coding for the protein CmWIP1 , and the presence of mutation (s) is sought in the sequence of the target gene, by comparison with the sequence of the non-mutated target gene. Then, the mutated plants are selected in the sequence of the gene coding for the target gene, here the gene coding for the protein CmWIP1.
  • step b) the DNA extracted from several M2 plants, for example 20 M2 plants, is first mixed and detection of mutations in the sequence of the target gene is carried out on the mixing (“pool”) of extracted DNA in order to reduce the number of mutation detection steps that must be carried out.
  • a step of amplifying the target sequences is carried out by PCR, using appropriate nucleic primers and the amplicons which are generated are heated, then cooled in order to generate DNA heteroduplexes between the DNA originating from a non-plant. mutated on the target nucleic acid and the DNA originating from a plant mutated on the target nucleic acid.
  • the DNA heteroduplexes are incubated in the presence of a cutting endonuclease at the level of the mismatches. Then the cleaved heteroduplexes are denatured and separated. Then the separated DNA strands are subjected to the mutation detection step (s) proper, for example by electrophoresis or else by HPLC under denaturing conditions (DHPLC).
  • s mutation detection step
  • HPLC HPLC under denaturing conditions
  • step b) the detection of mutation (s) in the target gene is carried out by the technique of HPLC under denaturing conditions (“DHPLC”), as described for example by Mc Callum et al. (2000, Plant Physiol., Vol. 123: 439-442 ).
  • HPLC HPLC under denaturing conditions
  • step c) from the plants mutated on the target gene, here the plants mutated in the gene coding for the protein CmWIP1 are selected, the plants which express the phenotype associated with the allele (g) of the genetic element (G / g).
  • the Tilling technique can be used, according to the present invention, for obtaining transformed plants comprising any one of the combinations of alleles (G / g) and (A / a ) described in the present description.
  • step b plants (i) having a mutation or more than one mutation in the gene coding for the protein CmWIP1 and (ii) having a mutation or more than one mutation in the gene encoding the ACS protein.
  • step c) one then selects, from the plants mutated on the two target genes, that is to say the plants mutated in the gene coding for the protein CmWIP1 and in the gene coding for the protein ACS, plants that express both the phenotype associated with the allele (g) of the genetic element (G / g) and the phenotype associated with the allele (a) of the genetic element (A / a) .
  • the present invention also relates to dicotyledonous plants of modified floral type in the genome of which at least one mutation has been introduced into the gene coding for the protein CmWIP1.
  • the present invention also relates to dicotyledonous plants of modified floral type which have been artificially mutated in the sequence of the gene coding for the protein CmWIP1, said plants expressing the phenotype associated with the allele (g) of the genetic element (G / g).
  • the present invention also relates to dicotyledonous plants of modified floral type into the genome of which has been introduced (i) at least one mutation in the gene coding for the protein CmWIP1 and (ii) at least one mutation in the gene coding for the protein ACS .
  • the present invention also relates to dicotyledonous plants of modified floral type which have been artificially mutated (i) in the sequence of the gene coding for the protein CmWIP1 and (ii) in the sequence of the gene coding for the protein ACS, said plants expressing at the times (i) the phenotype associated with the allele (g) of the genetic element (G / g) and the phenotype associated with the allele (a) of the genetic element (G / g).
  • the invention relates first of all to a method for obtaining a transformed plant aiming to insert the allele (G) in a plant not comprising this allele.
  • This type of process is particularly useful in that it makes it possible to insert the allele (G) into the genome of a plant, which will thus have a monoecious or andromonoic phenotype.
  • the subject of the invention is also a method of transforming plants aiming to suppress the allele (G) in a plant, or to replace the allele (G) with an allele (g) so as to obtain a plant of hermaphrodite phenotype or a female type plant.
  • the present invention also relates to dicotyledonous plants of modified floral type in the genome of which at least one mutation has been introduced into the gene coding for the protein CmWIP1.
  • the present invention also relates to dicotyledonous plants of modified floral type which have been artificially mutated in the sequence of the gene coding for the protein CmWIP1, said plants expressing the phenotype associated with the allele (g) of the genetic element (G / g).
  • the present invention also relates to dicotyledonous plants of modified floral type into the genome of which has been introduced (i) at least one mutation in the gene coding for the protein CmWIP1 and (ii) at least one mutation in the gene coding for the protein ACS .
  • the present invention also relates to dicotyledonous plants of modified floral type which have been artificially mutated (i) in the sequence of the gene coding for the protein CmWIP1 and (ii) in the sequence of the gene coding for the protein ACS, said plants expressing at the times (i) the phenotype associated with the allele (g) of the genetic element (G / g) and the phenotype associated with the allele (a) of the genetic element (G / g).
  • the invention relates first of all to a method for obtaining a transformed plant aiming to insert the allele (G) in a plant not comprising this allele.
  • This type of process is particularly useful in that it makes it possible to insert the allele (G) into the genome of a plant, which will thus have a monoecious or andromonoic phenotype.
  • the subject of the invention is also a method of transforming plants aiming to suppress the allele (G) in a plant, or to replace the allele (G) with an allele (g) so as to obtain a plant of hermaphrodite phenotype or a female type plant.
  • the method can also comprise a step d) of crossing plants obtained in step c) to obtain a bearing no more allele (G).
  • step a) consists in transforming a plant comprising the allele (G) in its genome, with a nucleic acid of “antisense” type as defined above, and selecting plants that no longer have the allele (G).
  • Such a nucleic acid of altered structure can consist of a regulatory polynucleotide (Pg), or a nucleic acid coding for a protein CmWIP1 altered in its sequence.
  • Pg regulatory polynucleotide
  • the method can also comprise a step d) of crossing plants obtained in step c) to obtain a bearing no more allele (G).
  • This type of process is particularly useful in that it makes it possible to obtain plants no longer comprising an allele (G), and which are of the hermaphrodite or gynoic type.
  • the invention also relates to a process for transforming plants aimed at replacing the allele (A) with the allele (a).
  • the method can also include a step d) of crossing plants obtained in step c) to obtain a no longer carrying allele (A).
  • the transformed plant according to the invention is characterized in that it has a genotype (aagg) and presents exclusively hermaphrodite flowers.
  • the polynucleotide (NG) when used comprises an inducible activating regulatory polynucleotide (PG).
  • the plants obtained according to the above process bear exclusively hermaphrodite flowers, and are therefore particularly advantageous from an industrial point of view, since they are not capable of self-pollination. These plants can therefore be used in processes for the creation of pure lines.
  • the transformation of plant cells can be carried out by various methods such as, for example, the transfer of the abovementioned vectors into plant protoplasts after incubation of the latter in a polyethylene glycol solution in the presence of divalent cations (Ca 2+), electroporation (Fromm et al. 1985), the use of a particle gun, or cytoplasmic or nuclear micro-injection (Neuhaus et al, 1987).
  • various methods such as, for example, the transfer of the abovementioned vectors into plant protoplasts after incubation of the latter in a polyethylene glycol solution in the presence of divalent cations (Ca 2+), electroporation (Fromm et al. 1985), the use of a particle gun, or cytoplasmic or nuclear micro-injection (Neuhaus et al, 1987).
  • One of the methods of transformation of plant cells which can be used in the context of the invention is the infection of plant cells by a bacterial cell host comprising the vector containing the sequence of interest.
  • the cell host can be Agrobacterium tumefaciens (An et al. 1986), or A. rhizogenes (Guerche et al. 1987).
  • the transformation of plant cells is carried out by the transfer of the T region of the extrachromosomal circular plasmid inducing Ti tumors of A. tumefaciens, using a binary system (Watson et al., 1994).
  • a binary system Wang et al., 1994.
  • two vectors are constructed. In one of these vectors, the T-DNA region was deleted, with the exception of the right and left edges, a marker gene being inserted between them to allow selection in plant cells.
  • the other partner in the binary system is a helper Ti plasmid, a modified plasmid which no longer has T-DNA but still contains the vir virulence genes necessary for the transformation of the plant cell. This plasmid is maintained in Agrobacterium.
  • the method described by Ishida et al. (1996) can be applied for the transformation of dicots.
  • the transformation is carried out according to the method described by Finer et al. (1992) using the tungsten or gold particle gun.
  • Cloning of the g locus based on the mapping was carried out by screening for the recombination elements in a population of 12,660 plants segregating for the g allele.
  • An F1 generation plant from the crossing of PI124112 (monoecious genotype AG-) x Gynadou (gynoic genotype A-gg) was backcrossed with Gynadou in order to generate an F2 generation population, for analysis purposes.
  • the g locus was located in an interval between the M261 and M365 markers on chromosome 4 (see figure 1A ).
  • the markers M261 and M365 are anchored in four different BAC clones coming from a genomic library of monoecious genotype, in order to generate a physical map of the locus.
  • this transposable element has a very high probability of being responsible for the phenotype of sex determination.
  • transposable elements are subject to epigenetic silencing inducing suppression of transposition and illegitimate genomic rearrangement
  • the methylation status of the DNA from the transposon to the g locus was examined, by PCR amplification sensitive to the McrBC endonuclease.
  • ORF3 which is located at a distance of less than 1 kb from the g locus, was specifically methylated in the gynoic genotype carrying the transposon ( figure 3A ).
  • the ORF3 open reading frame codes for a zinc finger transcription factor C2H2 and is homologous to members of the plant-specific WIP subfamily (Sagasser et al., 2002).
  • DNA methylation was also stronger in the first exon, the intron and near the 3'UTR sequence of the gynoic genotype, but to a lesser extent.
  • CmWIP1 the regulatory profiles of the mRNA of CmWIP1 were analyzed.
  • the expression levels of CmWIP1 were determined by quantitative PCR during sex determination and floral development.
  • the CmWIP1 gene has been shown to be strongly expressed in male flower buds at stage 6, and its expression decreases rapidly at later stages ( figure 5 ).
  • CmWIP1 mRNA was detected at a very low level, regardless of the stage of floral development.
  • the high and transient expression of CmWIP1 in male flower buds at the early stage of reproductive development coincides with the arrest of carpel development which has been described in recent work in cucurbits (Hao et al., 2003 ; Bai et al., 2004).
  • the localization of mRNA of CmWlP1 was found to be strongly confined in the fourth spiral of the male flower buds. This location corresponds to the primary carpel which will stop developing at the immediately later stage, in male flowers.
  • CmWIP1 The expression levels of CmWIP1 were also very low during the stages of development linked to sex determination (up to stage 6) in hermaphrodite flower buds (aagg) and in female flower buds of monoecious genotype ( figure 6 ). In other words, the expression of CmWIP1 at the early stage of floral development was always weak in the flowers which will develop into mature female structure, in all genotypes. All of these results strongly suggest that CmWlP1 has a role in determining the sex of melons, in particular in stopping the development of carpels in male flower buds.
  • Example 4 Determination of the floral type under the control of the element (G / g)
  • TILLING for “Targeted Induced Local-scale Lesions in Genome”
  • AG- monoecious genotype
  • EMS ethylmethanesulfonate
  • CmWIP1 is composed of two different domains, respectively a specific N-terminal domain and a C-terminal domain conserved within the zinc finger WIP proteins.
  • the floral phenotypes were observed in the homozygous M2 mutant plants, in comparison with the homozygous wild type plants of the same EMS family.
  • the three mutant alleles showed a redevelopment of the carpels, compared to male wild type flowers ( figure 7 ).
  • the mutants P193L, and S306F have become completely gynoic, which confirms that CmWIP1 is the gene for gynoecia.
  • the L77F mutant is a weak mutant.
  • Example 5 Spatial and temporal expression of the genetic control element (A / a)
  • in situ hybridizations were carried out using probes specific for the allele A on plants, and more precisely on floral meristems of male plants, females and hermaphrodites, of genotype AA GG , aa GG , AA gg and aa gg.
  • floral meristems A expression is locally strong and the hybridization signal is specifically detected in the primordia of carpels of female flowers and hermaphrodites of monoecious, andromonoic, gynoic and hermaphroditic plants.
  • the potential effects of the A / a gene, and of the ACS protein on the floral sexual phenotype and the architecture of the flower of plants not belonging to cucurbits were studied by transformation of Arabidopsis thaliana by Agrobacterium.
  • the transgenic Arabidopsis plants carrying the melon A allele or a have a phenotype in terms of floral architecture and siliques. (( Figures 9A and 9B ). Indeed, the pods of the Arabidopsis transformants are shorter than those of a wild Arabidopsis plant and the architecture of the flowers of the Arabidopsis transformants is very affected.
  • a person skilled in the art can also express the protein CmACS-7 in the carpel of a plant, preferably a cucurbitaceae to block the development of stamens or on the contrary inhibit the expression of the protein CmACS-7 in the carpel to promote the development of stamens. So the combination of active protein expression or mutants of CmACS-7 and CmWIP1 will make it possible to generate the different floral types described in TABLE 1.

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Claims (21)

  1. Verwendung einer Kombination von zwei genetischen Elementen zur Kontrolle der Entwicklung des Blütentyps einer zweikeimblättrigen Pflanze, wobei die Kombination jeweils umfasst:
    a) ein erstes genetisches Kontrollelement (A/a), das in der zweikeimblättrigen Pflanze in Form eines dominanten Allels (A) und eines rezessiven Allels (a) vorhanden ist, wobei:
    - das dominante Allel (A) aus einer Nukleinsäure (NA) besteht, die die Expression des Proteins ACS (Aminocyclopropancarboxylatsynthase) der Sequenz SEQ ID Nr. 3 gestattet,
    - das rezessive Allel (a) sich von dem dominanten Allel dadurch unterscheidet, dass das rezessive Allel (a) in der zweikeimblättrigen Pflanze nicht funktionell ist, und
    b) ein zweites genetisches Kontrollelement (G/g), das in der zweikeimblättrigen Pflanze in Form eines dominanten Allels (G) und eines rezessiven Allels (g) vorhanden ist, wobei:
    - das dominante Allel (G) aus einer Nukleinsäure (NG) besteht, die die Expression des Proteins CmWIPI ("C. melo-Zinkfingerprotein") der Sequenz SEQ ID Nr. 12 gestattet,
    - das rezessive Allel (g) sich von dem dominanten Allel dadurch unterscheidet, dass das rezessive Allel (g) in der zweikeimblättrigen Pflanze nicht funktionell ist, wobei selbstverständlich sein sollte, dass mindestens das zweite genetische Kontrollelement künstlich in die zweikeimblättrige Pflanze eingeführt worden ist.
  2. Verwendung einer Kombination von zwei genetischen Elementen nach Anspruch 1, dadurch gekennzeichnet, dass:
    - das aus einer Nukleinsäure (NA) bestehende dominante Allel (A) Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (PA), das in einer zweikeimblättrigen Pflanze funktionell ist, und
    (ii) eine Nukleinsäure, deren Expression durch das regulatorische Polynukleotid (PA) reguliert wird, wobei diese Nukleinsäure für das Protein ACS (Aminocyclopropancarboxylatsynthase) der Sequenz SEQ ID Nr. 3 codiert,
    - das nicht funktionelle rezessive Allel (a) sich von dem dominanten Allel durch eine nicht funktionelle Nukleinsäure unterscheidet und Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (Pa), dessen Nukleotidsequenz eine Insertion, eine Substitution oder eine Deletion in Bezug auf die Nukleinsäure umfasst, die vom Nukleotid 1 bis zum Nukleotid 5907 der Sequenz SEQ ID Nr. 1 reicht, und zu einer verringerten oder nicht vorhandenen Expression des Proteins ACS der Sequenz SEQ ID Nr. 3 führt,
    (ii) eine Nukleinsäure (Na), die für ein Protein codiert, das sich von dem Protein ACS der Sequenz SEQ ID Nr. 3 durch Substitution, Insertion oder Deletion einer oder mehrerer Aminosäuren unterscheidet und das nicht die biologische Aktivität des Proteins ACS in der zweikeimblättrigen Pflanze besitzt, und
    - das aus einer Nukleinsäure (NG) bestehende dominante Allel (G) Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (PG), das in einer zweikeimblättrigen Pflanze funktionell ist, und
    (ii) eine Nukleinsäure, deren Expression durch das regulatorische Polynukleotid (PG) reguliert wird, wobei die Nukleinsäure für die Expression des Proteins CmWIPI ("C. melo-Zinkfingerprotein") der Sequenz SEQ ID Nr. 12 codiert,
    - das nicht funktionelle rezessive Allel (g) sich von dem dominanten Allel (G) durch eine nicht funktionelle Nukleinsäure unterscheidet und Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (Pg), dessen Nukleotidsequenz mindestens eine Mutation, eine Insertion oder eine Deletion in Bezug auf die Nukleinsäure der Sequenz SEQ ID Nr. 13 umfasst, wobei diese Nukleinsäure zu einer verringerten oder nicht vorhandenen Expression des Proteins CmWIPI der Sequenz SEQ ID Nr. 12 führt, oder
    (ii) eine Nukleinsäure (Ng), die für ein Protein codiert, das sich von dem Protein CmWIPI der Sequenz SEQ ID Nr. 12 durch Substitution, Insertion oder Deletion einer oder mehrerer Aminosäuren unterscheidet und das nicht die biologische Aktivität des Proteins CmWIPI in der zweikeimblättrigen Pflanze besitzt.
  3. Verwendung einer Kombination von zwei genetischen Elementen nach einem der Ansprüche 1 und 2, dadurch gekennzeichnet, dass die Nukleinsäure, die für das Protein CmWIPI codiert, vom 5'-Ende zum 3'-Ende mindestens Folgendes umfasst:
    (i) eine Sequenz mit mindestens 95% Identität mit dem Polynukleotid, das vom Nukleotid 3000 bis zum Nukleotid 3617 der Sequenz SEQ ID Nr. 10 reicht, und
    (ii) eine Sequenz mit mindestens 95% Identität mit dem Polynukleotid, das vom Nukleotid 5458 bis zum Nukleotid 5901 der Sequenz SEQ ID Nr. 10 reicht.
  4. Verwendung einer Kombination von zwei genetischen Elementen nach einem der Ansprüche 2 und 3, dadurch gekennzeichnet, dass das regulatorische Polynukleotid (PG) die Nukleotidsequenz SEQ ID Nr. 11 umfasst.
  5. Nukleinsäure, umfassend vom 5'-Ende zum 3'-Ende mindestens:
    (i) eine Sequenz mit mindestens 98,5% Identität mit dem Polynukleotid, das vom Nukleotid 3000 bis zum Nukleotid 3617 der Sequenz SEQ ID Nr. 10 reicht, und
    (ii) eine Sequenz mit mindestens 99,5% Identität mit dem Polynukleotid, das vom Nukleotid 5458 bis zum Nukleotid 5901 der Sequenz SEQ ID Nr. 10 reicht.
  6. Nukleinsäure in Form des Allels (G) nach Anspruch 5 der Sequenz SEQ ID Nr. 10.
  7. Nukleinsäure, umfassend eine Nukleotidsequenz, die vom Nukleotid 3000 bis zum Nukleotid 5901 der Sequenz SEQ ID Nr. 10 reicht.
  8. Nukleinsäure, umfassend eine Nukleotidsequenz, die mindestens eine Veränderung, ausgewählt aus einer Mutation, einer Insertion oder einer Deletion, in Bezug auf die Nukleinsäure trägt, die vom Nukleotid 1 bis zum Nukleotid 2999 der Sequenz SEQ ID Nr. 10 reicht, wobei die veränderte Nukleinsäure zu einer veränderten Expression des Proteins CmWIPI der Sequenz SEQ ID Nr. 12, wenn sie die Expression dieses Proteins kontrolliert, im Vergleich zur Expression des Proteins CmWIPI, die von der Nukleinsäure kontrolliert wird, die vom Nukleotid 1 bis zum Nukleotid 2999 der Sequenz SEQ ID Nr. 10 reicht, führt.
  9. Rekombinanter Vektor, umfassend eine Nukleinsäure zum künstlichen Einführen in eine zweikeimblättrige Pflanze, wobei die Nukleinsäure aus den folgenden ausgewählt ist:
    a) einem genetischen Kontrollelement (G/g),
    wobei das genetische Kontrollelement (G/g) in Form von einem dominanten Allel (G) oder einem rezessiven Allel (g) vorliegt:
    - dem aus einer Nukleinsäure (NG) bestehenden dominanten Allel (G), das Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (PG), das in einer zweikeimblättrigen Pflanze funktionell ist, und
    (ii) eine Nukleinsäure, deren Expression durch das regulatorische Polynukleotid (PG) reguliert wird, wobei diese Nukleinsäure für die Expression des Proteins CmWIPI ("C. melo-Zinkfingerprotein") der Sequenz SEQ ID Nr. 12 codiert,
    - dem nicht funktionellen rezessiven Allel (g), das sich von dem dominanten Allel (G) durch eine nicht funktionelle Nukleinsäure unterscheidet und Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (Pg), dessen Nukleotidsequenz mindestens eine Mutation, eine Insertion oder eine Deletion in Bezug auf die Nukleinsäure der Sequenz SEQ ID Nr. 13 umfasst, wobei diese Nukleinsäure zu einer verringerten oder nicht vorhandenen Expression des Proteins CmWIPI der Sequenz SEQ ID Nr. 12 führt, oder
    (ii) eine Nukleinsäure (Ng), die für ein Protein codiert, das sich von dem Protein CmWIPI der Sequenz SEQ ID Nr. 12 durch Substitution, Insertion oder Deletion einer oder mehrerer Aminosäuren unterscheidet und das nicht die biologische Aktivität des Proteins CmWIPI in der zweikeimblättrigen Pflanze besitzt,
    oder
    b) einer Nukleinsäure nach einem der Ansprüche 5 bis 8.
  10. Wirtszelle, transformiert mit einem rekombinanten Vektor nach Anspruch 9, wobei die Wirtszelle in ihrem Genom eine künstlich durch den rekombinanten Vektor eingeführte Nukleinsäure umfasst, wobei die Nukleinsäure aus den folgenden ausgewählt ist:
    a) einem genetischen Kontrollelement (G/g),
    wobei das genetische Kontrollelement (G/g) in Form von einem dominanten Allel (G) oder einem rezessiven Allel (g) vorliegt:
    - dem aus einer Nukleinsäure (NG) bestehenden dominanten Allel (G), das Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (PG), das in einer zweikeimblättrigen Pflanze funktionell ist, und
    (ii) eine Nukleinsäure, deren Expression durch das regulatorische Polynukleotid (PG) reguliert wird, wobei die Nukleinsäure für die Expression des Proteins CmWIPI ("C. melo-Zinkfingerprotein") der Sequenz SEQ ID Nr. 12 codiert,
    - dem nicht funktionellen rezessiven Allel (g), das sich von dem dominanten Allel (G) durch eine nicht funktionelle Nukleinsäure unterscheidet und Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (Pg), dessen Nukleotidsequenz mindestens eine Mutation, eine Insertion oder eine Deletion in Bezug auf die Nukleinsäure der Sequenz SEQ ID Nr. 13 umfasst, wobei diese Nukleinsäure zu einer verringerten oder nicht vorhandenen Expression des Proteins CmWIPI der Sequenz SEQ ID Nr. 12 führt, oder
    (ii) eine Nukleinsäure (Ng), die für ein Protein codiert, das sich von dem Protein CmWIPI der Sequenz SEQ ID Nr. 12 durch Substitution, Insertion oder Deletion einer oder mehrerer Aminosäuren unterscheidet und das nicht die biologische Aktivität des Proteins CmWIPI in der zweikeimblättrigen Pflanze besitzt, oder
    b) einer Nukleinsäure nach einem der Ansprüche 5 bis 8.
  11. Wirtszelle nach Anspruch 10, dadurch gekennzeichnet, dass es sich um eine Zelle einer Pflanze aus der Familie Cucurbitaceae und vorzugsweise der Spezies Cucumis melo handelt.
  12. Pflanze aus der Familie Cucurbitaceae, transformiert mit einem rekombinanten Vektor nach Anspruch 9, wobei die Pflanze in ihrem Genom eine künstlich durch den rekombinanten Vektor eingeführte Nukleinsäure umfasst, wobei die Nukleinsäure aus den folgenden ausgewählt ist:
    a) einem genetischen Kontrollelement (G/g),
    wobei das genetische Kontrollelement (G/g) in Form von einem dominanten Allel (G) oder einem rezessiven Allel (g) vorliegt:
    - dem aus einer Nukleinsäure (NG) bestehenden dominanten Allel (G), das Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (PG), das in einer zweikeimblättrigen Pflanze funktionell ist, und
    (ii) eine Nukleinsäure, deren Expression durch das regulatorische Polynukleotid (PG) reguliert wird, wobei die Nukleinsäure für die Expression des Proteins CmWIPI ("C. melo-Zinkfingerprotein") der Sequenz SEQ ID Nr. 12 codiert,
    - dem nicht funktionellen rezessiven Allel (g), das sich von dem dominanten Allel (G) durch eine nicht funktionelle Nukleinsäure unterscheidet und Folgendes umfasst:
    (i) ein regulatorisches Polynukleotid (Pg), dessen Nukleotidsequenz mindestens eine Mutation, eine Insertion oder eine Deletion in Bezug auf die Nukleinsäure der Sequenz SEQ ID Nr. 13 umfasst, wobei diese Nukleinsäure zu einer verringerten oder nicht vorhandenen Expression des Proteins CmWIPI der Sequenz SEQ ID Nr. 12 führt, oder
    (ii) eine Nukleinsäure (Ng), die für ein Protein codiert, das sich von dem Protein CmWIPI der Sequenz SEQ ID Nr. 12 durch Substitution, Insertion oder Deletion einer oder mehrerer Aminosäuren unterscheidet und das nicht die biologische Aktivität des Proteins CmWIPI in der zweikeimblättrigen Pflanze besitzt, oder
    b) einer Nukleinsäure nach einem der Ansprüche 5 bis 8.
  13. Transformierte Pflanze nach Anspruch 12, dadurch gekennzeichnet, dass sie mindestens ein Allel (G) nach Anspruch 1 umfasst.
  14. Wirtszelle, transformiert mit:
    (i) einem rekombinanten Vektor, umfassend eine Nukleinsäure, ausgewählt aus:
    - einer Nukleinsäure, die ein Allel A oder a bestimmt, nach Anspruch 1,
    - einer Nukleinsäure, die vom 5'-Ende zum 3'-Ende mindestens Folgendes umfasst:
    (i) eine Sequenz mit mindestens 95% Identität mit dem Polynukleotid, das vom Nukleotid 5907 bis zum Nukleotid 6086 der Sequenz SEQ ID Nr. 1 reicht,
    (ii) eine Sequenz mit mindestens 95% Identität mit dem Polynukleotid, das vom Nukleotid 6181 bis zum Nukleotid 6467 der Sequenz SEQ ID Nr. 1 reicht, und
    (iii) eine Sequenz mit mindestens 95% Identität mit dem Polynukleotid, das vom Nukleotid 7046 bis zum Nukleotid 7915 der Sequenz SEQ ID Nr. 1 reicht,
    - einer Nukleinsäure in Form des Allels (A) nach Anspruch 2 der Sequenz SEQ ID Nr. 1,
    - einer Nukleinsäure in Form des Allels (a) nach Anspruch 2 der Sequenz SEQ ID Nr. 2,
    - einer Nukleinsäure, umfassend eine Nukleotidsequenz, die vom Nukleotid 1 bis zum Nukleotid 5906 der Sequenz SEQ ID Nr. 1 reicht,
    - einer Nukleinsäure, umfassend wobei eine Nukleotidsequenz, die mindestens eine Veränderung, ausgewählt aus einer Mutation, einer Insertion oder einer Deletion, in Bezug auf die Nukleinsäure trägt, die vom Nukleotid 1 bis zum Nukleotid 5907 der Sequenz SEQ ID Nr. 1 reicht, wobei die veränderte Nukleinsäure zu einer veränderten Expression des Proteins ACS der Sequenz SEQ ID Nr. 3, wenn sie die Expression dieses Proteins kontrolliert, im Vergleich zur Expression des Proteins ACS, das von der Nukleinsäure kontrolliert wird, die vom Nukleotid 1 bis zum Nukleotid 5907 der Sequenz SEQ ID Nr. 1 reicht, führt, und
    - einer Nukleinsäure, umfassend eine Sequenz, die vom Nukleotid 1 bis zum Nukleotid 3650 der Sequenz SEQ ID Nr. 2 reicht, und
    (ii) einem rekombinanten Vektor nach Anspruch 9,
    wobei die Wirtszelle in ihrem Genom eine künstlich durch den rekombinanten Vektor eingeführte Nukleinsäure umfasst.
  15. Wirtszelle nach Anspruch 14, dadurch gekennzeichnet, dass es sich um eine Zelle einer Pflanze aus der Familie Cucurbitaceae und vorzugsweise der Spezies Cucumis melo handelt.
  16. Nukleinsäure, verwendbar als Primer, die spezifisch mit einer Nukleinsäure nach einem der Ansprüche 5 bis 8 hybridisiert.
  17. Verfahren zum Nachweis des Vorhandenseins eines Allels (G) oder (g) nach Anspruch 1, wobei das Verfahren die folgenden Schritte umfasst:
    1) Inkontaktbringen einer Nukleotidsonde oder einer Mehrzahl von Nukleotidsonden, die spezifisch mit einer Nukleinsäure nach einem der Ansprüche 5 bis 8 hybridisiert/hybridisieren, mit der zu testenden Probe;
    2) Nachweisen des gegebenenfalls gebildeten Komplexes zwischen der Sonde bzw. den Sonden und der in der Probe vorhandenen Nukleinsäure.
  18. Verfahren zum Erhalten von Pflanzen, die künstlich in dem für das Protein CmWIPI codierenden Gen mutiert sind, umfassend die folgenden Schritte:
    a) Erzeugen einer Sammlung von mutierten zweikeimblättrigen Pflanzen durch chemische Mutagenese;
    b) Selektieren, aus der im Schritt a) erzeugten Sammlung von mutierten Pflanzen, derjenigen Pflanzen, die eine Mutation oder mehr als eine Mutation in dem für das Protein CmWIPI codierenden Gen besitzen.
    c) Selektieren, unter den im Schritt b) selektierten mutierten Pflanzen, derjenigen Pflanzen, die den Phänotyp exprimieren, der mit dem Allel (g) des genetischen Elements (G/g) nach Anspruch 1 assoziiert ist.
  19. Verfahren nach Anspruch 18, dadurch gekennzeichnet, dass die Pflanzen außerdem in dem für das Protein ACS codierenden Gen mutiert sind, und dadurch, dass:
    - man im Schritt b) diejenigen Pflanzen selektiert, die
    (i) eine Mutation oder mehr als eine Mutation in dem für das Protein CmWIPI codierenden Gen besitzen und
    (ii) eine Mutation oder mehr als eine Mutation in dem für das Protein ACS codierenden Gen besitzen,
    - man im Schritt c) unter den Pflanzen, die in dem für das Protein CmWIPI codierenden Gen und in dem für das Protein ACS codierenden Gen mutiert sind, diejenigen Pflanzen selektiert, die sowohl den mit dem Allel (g) des genetischen Elements (G/g) assoziierten Phänotyp als auch den mit dem Allel (a) des genetischen Elements (A/a) assoziierten Phänotyp exprimieren.
  20. Verfahren zum Erhalten einer transformierten Pflanze aus der Familie Cucurbitaceae, dadurch gekennzeichnet, dass es die folgenden Schritte umfasst:
    a) Transformation mindestens einer Pflanzenzelle einer Pflanze von Interesse, die nicht das Allel (G) nach Anspruch 1 in ihrem Genom umfasst, mit einer Nukleotidsequenz (NG) nach Anspruch 1 oder einem rekombinanten Vektor, umfassend eine solche Nukleinsäure;
    b) Selektion der im Schritt a) erhaltenen transformierten Zellen, die (NG) in ihre Nukleinsäure integriert haben;
    c) Regeneration einer transformierten Pflanze aus den im Schritt b) erhaltenen transformierten Zellen.
  21. Verfahren zum Erhalten einer transformierten Pflanze aus der Familie Cucurbitaceae, dadurch gekennzeichnet, dass es die folgenden Schritte umfasst:
    a) Transformation mindestens einer Pflanzenzelle einer Pflanze von Interesse, die das Allel (G) nach Anspruch 1 in ihrem Genom umfasst, mit einem nicht funktionellen regulatorischen Polynukleotid (Pg) oder mit einer Nukleinsäure (Ng), die für ein verändertes Protein CmWIP1 codiert, jeweils nach Anspruch 1, oder mit einer Antisense-Nukleinsäure (NG),
    b) Selektion transformierter Zellen, die aus einer im Schritt a) erhaltenen Pflanze stammen und in ihr Genom mindestens eine Kopie eines nicht funktionellen regulatorischen Polynukleotids (Pg) oder einer Nukleinsäure (Ng), die für ein verändertes Protein CmWIP1 codiert, nach Anspruch 1 integriert haben oder die das Allel (G) nicht mehr tragen,
    c) Regeneration einer transformierten Pflanze aus den im Schritt b) erhaltenen transformierten Zellen.
EP09740394.3A 2008-07-28 2009-07-27 Kombination von zwei genetischen elementen zur kontrolle der blütenentwicklung einer zweikeimblättrigen pflanze, deren anwendung bei nachweis- und selektionsverfahren Active EP2318536B1 (de)

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FR0855146A FR2934277B1 (fr) 2008-07-28 2008-07-28 Combinaison de deux elements genetiques pour le controle du developpement du type floral d'une plante dicotyledone, et mise en oeuvre dans des procedes de detection et de selection
PCT/FR2009/051510 WO2010012948A2 (fr) 2008-07-28 2009-07-27 Combinaison de deux elements genetiques pour le controle du developpement du type floral d'une plante dicotyledone, et mise en oeuvre dans des procedes de detection et de selection

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FR2985635A1 (fr) * 2012-01-16 2013-07-19 Inst Nat De Rech Agronomique Cucurbitacees androiques, procedes d'obtention et utilisations de ces cucurbitacees
CN103215289B (zh) * 2013-01-10 2014-06-25 北京市农林科学院 导致西瓜两性花发育的基因a序列及其获得方法
CN107385055A (zh) * 2017-08-09 2017-11-24 青岛科技大学 甜瓜单性花性状相关acs‑7基因的snp分子标记及应用
WO2020025133A1 (en) * 2018-08-01 2020-02-06 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Mutant allele of the aco2 gene
EP4079751A1 (de) 2021-04-22 2022-10-26 Institut national de recherche pour l'agriculture, l'alimentation et l'environnement Identifizierung eines neuen gens, das an der geschlechtsbestimmung bei cucurbitaceae beteiligt ist

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FR2900415B1 (fr) * 2006-04-28 2010-08-13 Agronomique Inst Nat Rech Systeme genetique pour le controle du developpement du type floral d'une plante dicotyledone, et mise en oeuvre dans les procedes de detection et de selection

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IL210908A (en) 2016-05-31
EP2318536A2 (de) 2011-05-11
WO2010012948A3 (fr) 2010-04-15
ES2798147T3 (es) 2020-12-09
CN102165067A (zh) 2011-08-24
US20110314569A1 (en) 2011-12-22
WO2010012948A2 (fr) 2010-02-04
PT2318536T (pt) 2020-06-18

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